Bioactive mono-dispersed nanospheres with long-term antibacterial effects for endodontic sealing

Abstract

Endodontic sealers with antibacterial capability play an important role in preventing reinfection of an endodontically treated root canal and improving the long-term success of root canal treatment. However, current endodontic sealers rapidly lose their antibacterial properties after fixation. In this work, we designed and synthesized quaternized mono-dispersed bioactive nanospheres as a potential substrate for the development of a long-term antibacterial endodontic sealer with excellent cytocompatibility and biocompatibility. First, mono-dispersed silica-based bioactive glass nanospheres (SBG-NS) were prepared via a modified sol-gel process. Next, a series of quaternary ammonium methacrylate salts (QAMs) with broad antibacterial spectra were synthesized and grafted onto the surfaces of the SBG-NS via a two-step coupling approach. The antibacterial effect of the quaternary ammonium polymethacrylate (QAPM)-containing SBG-NS (SBG-QAPM) against persistent microorganisms associated with infected root canals was evaluated using a direct contact test. Evaluations of the SBG-QAPM cytocompatibility and biocompatibility were performed using LIVE/DEAD staining, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2, 5-tetrazoliumbromide (MTT) assay, and a calvarial implantation model. The results showed that the SBG-QAPMs had the strongest long-term antibacterial effect against the Enterococcus faecalis, Streptococcus mutans, and Streptococcus sanguis during the study period, the best cytocompatibility, and the lowest systemic inflammation compared to three commercial products: ProRoot MTA, Endomethasone C, and AH Plus. In addition, the SBG-QAPMs showed excellent stability in aqueous solution. This work indicates that the SBG-QAPMs are promising substrates for the development of long-term antibacterial endodontic sealers.

Fig. 1

Characterizations of SBG-NS. (A) SEM image of SBG-NS, showing that the SBG-NS were spherical and mono-dispersed. (B) Size distribution of the SBG-NS.

Fig. 2

Characterizations of the QAMs. (A) ¹H NMR spectrum of the HAEMB monomer. (B) ATR-FTIR spectra of the four QAMs (PAEMB, NAEMB, HAEMB, and OAEMB).

Fig. 3

(A) The ATR-FTIR spectra of SBG-NS, SBG-NS/KH-570, and SBG-QAPMs (SBG-PA, SBG-NA, SBG-HA, and SBG-OA). (B) EDS spectra of the SBG-NS, SBG-NS/KH-570, HAEMB, and SBG-HA. (C) The elemental analyses of SBG-NS, SBG-NS/KH-570, and SBG-QAPMs. **P < 0.01, ***P < 0.001. (D) SEM image of SBG-HA. Scale bar = 500 nm. (E) Size distribution of the SBG-HA.

Fig. 4

(A) The coupling ratio of KH-570 to SBG-NS. (B) The grafting ratio of QAMs (PAEMB, NAEMB, HAEMB, and OAEMB) to SBG-NS/KH-570 (B). ***P < 0.001. (C) The stability of the SBG-QAPMs in PBS solution.

Fig. 5

Antibacterial effect of the SBG-QAPMs (SBG-PA, SBG-NA, SBG-HA, and SBG-OA) and three commercial endodontic products (Endomethasone C, AH Plus, and ProRoot MTA) against the E. faecalis, S. mutans, and S. sanguis. (+), bacterial suspension only; (−), culture medium only.

Fig. 6

In vitro cytocompatibility (LIVE/DEAD staining) of the SBG-HA to the PDLSCs compared with Endomethasone C, AH Plus, and ProRoot MTA. The green fluorescence represented viable cells while the red fluorescence represented dead cells. Scale bar = 500 µm.

Fig. 7

MTT assay of the SBG-HA and Endomethasone C, AH Plus, and ProRoot MTA. **P < 0.01, ***P < 0.001.

Fig. 8

In vivo inflammatory reaction of the SBG-HA, Endomethasone C, AH Plus, and ProRootMTA after being implanted in a calvarial defect of SD rats for 2 and 4 weeks. *P < 0.05, **P < 0.01, ***P < 0.001.

Scheme 1

Schematic diagram of synthesizing SBG-QAPMs.